Find answers to some of the most frequently asked questions about our Instruments & Software, Cell Lysis, ChIP, FFPE Nucleic Acid Extraction, and Nucleic Acid Shearing.
While some denaturing is tolerable, I have disulfide bonds in my protein that I do not want to disrupt. Will truXTRAC Protein Extraction Buffer DF leave disulfide bonds intact?
I am interested in extracting intact proteins for downstream functional assays. Functional assays involve a co-factor stain that can be used to check for protein presence by SDS-PAGE and if protein is strep tagged, it can be detected by anti-strep antibody on Western blot. Will truXTRAC® Protein Extraction Buffer SuperB affect this functional assay?
What non-ionic detergents work well with AFA?
- Triton X-100
- Octylglucoside (octyl-beta-D-glucopyranoside)
Does Covaris provide an alternative type of detergent containing a buffer for LC/MS analysis?
What is the best buffer to increase total protein yields?
Describe the most suitable buffer for recovering proteins in native conformation?
What is the best buffer for extracting denatured proteins?
Can I take extracted protein in Buffer TP or Buffer MJ for direct analysis by MS?
Do native protein extraction buffers have any percent of organic compounds?
What is the advantage of using truXTRAC Protein Extraction Buffer SuperB instead of truXTRAC Protein Extraction Buffer N?
How should I collect adherent cells and perform sample quantification?
Why do I need to run a fixation time course? What cell types is it most critical for and why?
Why is the methanol-free formaldehyde provided with truChIP kits recommended over other fixing solutions?
Why is the fixation time in the Covaris truChIP ® protocol so short?
Why do we need to run a shearing time course if we have already done ChlP before?
What type of cells can truChIP kit be used for?
What are the lowest cell inputs that have been tested?
I want to process 50M cells/sample–is it possible?
Does Covaris provide a protocol for tissue ChIP?
Is it possible to fragment native ChIP using a Covaris?
Can you simultaneously lyse and fragment in one tube?
May I use my own buffers and reagents?
May I combine your protocol with my other commercial protocols?
How does having reduced SDS concentrations help with the Covaris kit?
Is there a required RNase treatment?
Why am I not seeing a gradual reduction in fragment size distribution as I shear my samples?
There are a few possibilities why no shearing is observed:
- The cells were processed using reagents and protocols not optimized for use with AFA
- The cells are being severely over-fixed
- The crosslinked sheared chromatin has not been reversed or purified properly for DNA size range analysis
I optimized protocols for a cell line and now am observing inconsistent shearing results. Why?
Why did my IP fail?
IP can fail for a myriad of reasons. Some of the most common ones are:
- Over shearing resulting in epitope damage
- Fixation was not optimized for the protein of interest–the crosslinking was insufficient
- Incorrect IP buffer that has not been optimized for use with the selected antibody
Is it true that the fixation time cannot be reduced if a target of interest is not abundant?
I have a large protein complex. What methods are recommended for these sample types?
If I fix it in less/time, will I lose some downstream sensitivity?
What is the average processing time per sample using truChIP?
Why do I need to control the temperature?
Specifically, how does the duty factor (DF), power, and duration impact the fragment length?
May I use the truChIP kit with the M220 focused ultrasonicator?
Are larger processing volumes available?
What settings should I use on the Covaris for ChIRP/Hi-C, etc.?
Where can I find protocols for the cryoPREP® Extraction Systems?
What impact levels are recommended with cryoPREP (CP02) Impactor for my tissue samples?
Impact level varies and will depend on both the tissue type and input mass. Typically, Covaris recommends using level 5 or 6 for hard tissues such as skin, blood vessels, cornea, retina, cartilage, and bone. We recommend using level 2 to 4 if processing soft tissues.
For more information on the recommended impact levels, refer to the chart below:
|20-80 mg||80-300 mg||300-500 mg||500-1000 mg||1000-2000 mg|
Can I impact the sample in the tissueTUBE more than once?
What is the working mass range of the different tissueTUBEs?
Please refer to the table below for the recommended impact levels according to their mass ranges:
|20-80 mg||80-300 mg||300-500 mg||500-1000 mg||1000-2000 mg|
Can I add a buffer to my tissue in the tissueTUBE before processing with the cryoPREP CP02 Impactor?
If residual tissue remains on the walls of the tissueTUBE after transferring the pulverized material, what buffer is recommended to collect remaining material from the tissueTUBE for nucleic acid extraction?
If residual tissue remains on the walls of the tissueTUBE after transferring the pulverized material, what buffer can I use to transfer from tissueTUBE for protein extraction?
Can cryoPREP Extraction Systems be used for plant material?
Once the sample is pulverized, is it mandatory for the sample to be processed immediately, or can it be stored?
Can cryofractured tissue be freeze/thawed multiple times?
Can the samples in the tissueTUBE thaw?
Does the frozen tissue after cryo-impact still remain frozen through the whole process of fracturing?
Does the process result in lysed/fractured cells or is it possible to fix the resulting frozen powder in formaldehyde to recover intact cells for FACS sorting?
What are the reasons why the cryofractured material is not completely pulverized?
Certain tissues such as high adipose content tissues will “cake” immediately after impact rather than cryofracturing into distinct powder. Therefore, we recommend for the “caked tissue” to be quickly disrupted into smaller pieces inside the tissueTUBE prior to transfer into the transfer tube. Some other reasons for inefficient cryofracturing are:
- Tissue mass is too large for the tissueTUBE being used, or the impact level is too low for the tissue mass being used. Please refer to the impact level and tissue mass chart.
- Tissue is not centered in the tissueTUBE during impact.
- Liquid nitrogen incubation time is too short. We recommend a minimum of 30 seconds for tissue masses less than 100 mg, and 60 seconds for tissue mass of up to 2 g.
Can cryoPREP CP02 Impactor impact pig skin (0.5mm thickness)?
Why has the tissueTUBE broken open during cryofracture?
There are various reasons why the tissue TUBES break open, these include:
- Tissue mass was too large for the tissueTUBE being used
- Tissue was not centered in the tissueTUBE. Tissues expand during impact, and if placed close to the seams, it will burst the seams of the tissueTUBE.
- Tissue was not properly flash frozen in liquid nitrogen. The key is to submerge the sample in liquid nitrogen for 30 to 60 seconds depending on the mass of the tissue.
- Previously frozen tissues will have sharp edges which can pierce through the tissueTUBEs. In such cases, we recommend an initial low impact level of 1 or 2, followed by another treatment with liquid nitrogen, and another impact at 4-6 depending on the tissue type.
- When processing tissues in tissueTUBEs TT05 and TT1, it is important to loosen the transfer tube by a quarter turn to allow venting of the rapidly expanding liquefied air.
What is the cryoPREP (CP02) Impactor and its key benefits?
What accessories and/or consumables do I need to process my tissue samples?
When would I use standard tissueTUBE or extra thick tissueTUBE (XT)?
Can I store my samples in tissueTUBE?
At what temperature can the tissueTUBEs be stored?
What material is the tissueTUBE material made of?
What is the dimension of a piece after pulverization?
Can cryofractured tissue be stored at -80°C in the glass Covaris transfer tubes?
On what AFA instruments can the cryofractured samples be treated for biomolecules extraction?
Are service plans for the cryoPREP (CP02) Impactor offered?
Where can I find protocols for FFPE nucleic acid extraction?
Can I extract RNA or total nucleic acids using truXTRAC FFPE kits?
Can I extract proteins using truXTRAC FFPE kits?
Are the truXTRAC FFPE kits xylene-free?
What tissue sample types can be used?
What is the maximum sample size for use with FFPE truXTRAC protocols?
What collection tools are recommended for sampling slide-mounted FFPE sections?
Why is my DNA/RNA yield low? How can I improve my yield?
Low yield might be caused by several different factors such as:
- Low tissue to wax ratio in FFPE section: Excess paraffin will adversely affect the yield and quality of DNA and RNA extracted from FFPE tissue. We strongly advise trimming off any excess paraffin before sectioning a FFPE tissue block, or after the section has been cut from the FFPE block. A ratio of 80% tissue to 20% paraffin, or higher, is ideal. Repeat the procedure using additional sections until desired yield is achieved. In your initial use of the truXTRAC FFPE kit, use FFPE blocks that have been well characterized for yield and quality.
- Insufficient tissue input: Increase FFPE tissue section thickness or use more sections up to 5mg total weight.
- Proteinase K stored above recommended temperature or expired: Repeat the procedure using fresh Proteinase K. Always store Proteinase K solution at -20°C after resuspension.
If using bead purification, low DNA yield may also be caused by:
- Incorrect number of beads were added: Ensure beads are resuspended well by vortexing beads thoroughly before each use.
- Binding was incomplete: Make sure the correct reagent volumes were used and mixed well before the 56°C incubation.
- Beads lost during pipetting steps: Slowly draw up the supernatant into the pipette tip without disturbing the beads. It is normal for beads to stick to the pipette tips during the elution step without having an effect on the yield.
Why do I have no DNA/RNA?
The concentration of DNA/RNA is too low, what could be the cause?
When following the FFPE DNA protocol with bead purification I notice clumping of magnetic beads during wash steps. Is this a problem?
Magnetic beads are sticking to the pipette tips during elution whilst following the FFPE DNA bead purification protocol. Will this affect my DNA yield?
Why does my DNA not perform well in downstream applications such as qPCR?
The DNA fragment size is too large when following Option A of the FFPE DNA protocol. What is the cause of this?
Which Covaris Focused-ultrasonicators are compatible with truXTRAC FFPE protocols?
How do I convert a method developed on an S2 or E210 for use with SonoLab™ 7?
What happened to Intensity (as employed in previous versions of SonoLab)?
Why do I need to change the water in the tank every day?
Besides the daily changing of water in the bath, what are the other maintenance recommendations?
For all instruments, except the M220, the water bath and the degassing lines should be rinsed once a month with a 10% bleach solution. Refer to the recommendations below for more guidance:
- Fill the bath with 10% bleach solution
- Lower the transducer and run the degassing pump for a few minutes
- Raise the transducer and empty the tank
- Lower the transducer and run the pump for 5-10 seconds to empty the lines
- Fill the water bath with fresh water and repeat the procedure
The ME220 bleach protocol is run using the fill bottle based on the procedure from the user manual.
I have the Covaris Water Conditioning System. What kind of maintenance should I perform?
The Water Conditioning System (WCS) automatically circulates water through a particulate filter and ultraviolet (UV) sterilizer to ensure that water remains clean and free from algae growth for up to a month. When the system runs continuously, it maintains the proper water quality required for optimum performance of the transducer. Without the WCS, proper water quality is maintained by daily changes of the water in the bath. On a monthly basis, the water tank should be cleaned with a bleach solution as described above.
IMPORTANT: Disconnect the hoses to and from the WCS when performing the bleaching procedure. DO NOT circulate the bleach solution through the WCS. The UV lamp and filter should be replaced once a year as indicated by the hour meter on the front of the WCS. Instructions for this can be found in the user manual.
Why is it so important to degas the water in the tank?
Can I add algaecide to my water bath?
When I am done using the Covaris system, how should I properly shut down the instrument?
What is AFA-grade water?
The chiller is for some reason taking over an hour to chill the water bath on my S220. What could be the cause?
I have a VWR chiller that came with the Covaris instrument. I can’t change the temperature on the chiller control panel. What should I do?
How can I convert a method from one model instrument to another?
What is the latest version of SonoLab 6 software (for E and LE instruments)?
SonoLab 6.2.8 was the last version of SonoLab 6 software released. SonoLab 6 is technically obsolete and has been replaced by SonoLab 7.3. The SonoLab 7 platform features improved error recording and the addition of history files. The same settings are used for protocols in both versions of software. If you have questions or concerns about upgrading, please contact [email protected].
|6.2.8 (can be replaced by 188.8.131.52)||X||X|
Can AFA water be cooled in advance to speed up the preparation time?
How can I be sure that the degas pump is running?
Our facility is prohibited from using bleach. Is there an alternative that can be used?
If the maintenance or check flow light appears on my WCS, how do I turn this off?
The Check Flow light indicates that the pressure is building up and water flow is restricted. The hose connections may need to be reset if they are not seated properly. If this does not solve the problem, the filter may need to be replaced if it is blocked.
Where can I find DNA shearing protocols?
What are the DNA fragment sizes I can get using Covaris Focused-ultrasonicator instruments?
How can I shear genomic DNA into fragment sizes larger than 5 kb?
We have 300 bp DNA which we want to shear to 150 to 200 bp. Can we use the recommended protocol?
Our recommended protocols for shearing genomic DNA as a starting material requires more than a tenfold larger input starting material than the desired fragment size. When the difference between the size of DNA starting material and desired fragment size is small, increasing the AFA treatment time, but maintaining all other AFA parameters is recommended. For example, on the M220 instrument using a microTUBE-130 to shear gDNA to 150 bp, treatment time is 330 sec. If using 300 bp DNA as the starting material for the same size, you will need to increase the shearing time beyond 330 sec. Please run a time course with 10% incremental steps, and check the results on the Bioanalyzer.
Relationship between DNA fragment sizes and total energy needed.
I do not see any settings for the fragment size I am interested in, but I see settings for size ranges a bit over and a bit below what I am interested in. How can I optimize settings to get the size range of interest?
Why does the size distribution of fragments seem to get wider with increasing size range?
I have an Illumina protocol for creating 400bp fragments that uses T6 round bottom glass tubes. What alternatives are recommended?
Aside from the phosphodiester bonds, are there any other bonds broken during processing of my samples with the Covaris AFA?
In what buffer type can I shear my DNA?
Can I use the Covaris Focused-ultrasonicator instrument to fragment RNA? If so, what conditions should I use?
Can I use the published protocols to shear PCR amplicons?
Does the DNA source have an effect on the shearing conditions and results?
What is the minimum concentration of input DNA for the g-TUBE?
Can I use a microcentrifuge tube to shear the DNA?
Can I use microTUBE or miniTUBE for DNA storage?
I notice a fiber inside the microTUBE/milliTUBE. What is the purpose of this fiber, and should I be worried about contamination of my samples from this fiber during treatment?
Which centrifuges are compatible with the g-TUBE?
- Eppendorf MiniSpin plus
- Eppendorf Model 5415 R with temperature set at 30° C
- Eppendorf Model 5424
If you have another brand of centrifuge, please contact [email protected].
Why am I not getting consistent results when shearing DNA?
Inconsistent results might be caused by several different factors, however, there are few important things to check first:
- Sample volume: Each consumable has been optimized for a specific sample volume in order to allow optimal performance. For example, the presence of large headspace allows for the occasional formation of an air gap in the tubes, disrupting the consistent fragmentation of DNA to the desired size range. Click here to find instrument protocols which include instrument specific recommended sample vessels for different volumes.
- Water level: The water level is critical for DNA shearing. Not only does the water allow for the acoustic energy to couple from the transducer into the tube, it is also important in keeping your sample at the appropriate temperature during processing and minimizing vibrations which could lead to glass tube breakage. Exact water levels to follow are published for each instrument and consumable, click here for the latest information.
- Water bath temperature: Water temperature should be closely controlled and matched to the application. Warmer temperatures promote less forceful collapse of acoustic cavities within the sample fluid, causing a shift toward larger mean fragment size, therefore the water bath temperature during DNA shearing should be tightly controlled.
- Degas level of water: For S, E and LE- series instruments, insufficient degas levels within the bath may result in less efficient acoustic coupling and thereby shift the mean fragment size. System degas pumps should be run in advance and during AFA treatment. Prior to running a process the water bath should be degassed for at least 30 min to one hour, until the “degas” indicator in the SonoLab software can be checked without error.
- Water purity: Foreign materials such as algae and particulates may scatter the high frequency focused acoustic beam, resulting in a shift to larger mean fragment size. Bath water should be pure distilled or DI water, changed daily or cleansed by a Covaris Water Conditioning System.